Glia exhibit large fluctuations in intracellular calcium both spontaneously and in response to neuronal activity, although the influence of glial calcium oscillations on neuronal function is not well understood. By screening for temperature-sensitive seizure mutations in Drosophila we identified the zydeco mutation, which disrupts a glial-specific sodium-potassium dependent calcium exchanger (NCKX). Preliminary data indicates that NCKX is required in glia to regulate neuronal excitability, and that mutation of NCKX reduces the frequency of glial calcium oscillations. This proposal will investigate how glial calcium signaling acutely affects neuronal function. (1) We will characterize the basic properties of glial calcium oscillations in Drosophila by transgenically expressing a calcium sensitive green fluorescent protein in glia and determining the relationship between glial calcium and electrical activity in associated neurons. We will investigate how mutation of NCKX affects the magnitude and kinetics of calcium waves in glia. (2) We will perform a comprehensive analysis of synaptic transmission and plasticity at the Drosophila neuromuscular junction while manipulating glial calcium levels with a transgenic temperature controlled glial cation channel. We will also determine the consequence of NCKX mutation on neuronal function by analyzing synaptic transmission and neuronal excitability in zydeco mutants. These experiments will elucidate the mechanism by which alteration of a novel glial-neuronal signaling pathway mediated by calcium enhances excitability in the brain and predisposes to seizures. Public Health Relevance: Glial calcium waves have been associated with the generation of seizures in several mammalian models, but the relationship between glial calcium elevation and neuronal excitability is unclear. By characterizing the changes in glial and neuronal function in seizure-susceptible zydeco mutants, we will determine the role of glia in generating hyperactive neuronal networks, which may provide insights into the pathophysiology of seizures.